Drug screening model is an indispensible platform for drug activity research in the drug research and development. High Content Analysis or High Content Screening (HCS) is an automatic platform utilizing high-throughput fluorescence/bright field microscopic imaging and quantitative image analysis integrated with open reagents and fluorescence labeling technique, which can detect impact to cells by samples to be screened in various aspects, such as cellular morphology, growth, differentiation, migration, apoptosis, metabolic pathway and signal transduction simultaneously, obtain a lot of relevant information in a single experiment so as to determine the biological activity and potential toxicity while keeping the cell structure and function integral. It is an inevitable tendency for creative drug screening research to establish a cell model for screening kinase inhibitors and to combine it with high-throughput screening technique on the basis of High Content technique.
Kinase as target for drugs (such as anti-tumor drugs) has become a hotspot for the new drug development internationally. Macrophage colony stimulating factor receptor kinase (c-Fms, CSF-1R) is a product encoded by protooncogene c-fms, and belongs to the type ° C. tyrosine kinase family, as stem cell growth factor receptor(c-Kit), platelet-derived growth factor receptor (PDGFR), Fms-like tyrosine kinase 3 (Flt-3) and so on. After binding to macrophage colony stimulating factor (M-CSF, CSF-1), the tyrosine kinase activity of c-Fms is activated, promotes the survival, proliferation and proliferation of mononuclear macrophage cell line (Sherr C. J., Roussel M. F., Rettenmier C. W., Colony-stimulating factor-1 receptor (c-fms)[J]. J. Cell Biochem., 1988, 38(3):179-187). Factors such as repeated replication, mutation, chromosome translocation of C-fms gene and over-expression of M-CSF can abnormally enhance c-Fms kinase activity, result in abnormal proliferation of mononuclear macrophage or high expression of relevant inflammatory factors, and finally lead to the development of diseases such as hematopoietic system disorder, malignant tumors, inflammation and atherosclerosis.
C-Fms inhibitors, which can inhibit the phosphorylation of receptor kinases and block cell signal pathway mediated by receptor kinases, are candidate drugs for treating c-Fms-related diseases (Douglass T. G., Driggers L., Zhang J. G., et al., Macrophage colony-stimulating factor: not just for macrophages anymore! A gateway into complex biologies [J]. Int. Immunopharmacol, 2008, 8(10): 1354-1376). Signal transducer and transcription activator (STAT1) which are important molecules of cell signal downstream of c-Fms kinases, enter nucleus upon activation by c-Fms, and activate the transcription of relevant genes (Hamilton J. A., CSF-1 and cell cycle control in macrophages [J]. Mol. Reprod. Dev., 1997, 46(1): 19-23). The extent of STAT1 nuclear translocation directly reflects the kinase activity of c-Fms.
As reported in current documents, methods for screening c-Fms inhibitors mainly includes methods for determining kinase activity at the molecular level and methods of western blot for detecting kinase phosphorylation at cellular level, however, they have shortcomings such as complex and tedious operations and low quantification extent. In addition, since the phosphorylation of receptor kinases such as c-Fms occurs in a short time, it is difficult to detect it accurately. Furthermore, due to easy dimerization in vitro and the like, there is no report on cell models of high-throughput screening of c-Fms inhibitors based on High Content Analysis.